MPH1 is a thylakoid membrane protein involved in protecting photosystem II from photodamage in land plants

Photosystem II (PSII) is highly susceptible to photoinhibition caused by environmental stimuli such as high light; therefore plants have evolved multifaceted mechanisms to efficiently protect PSII from photodamage. We previously published data suggesting that Maintenance of PSII under High light 1 (MPH1, encoded by AT5G07020), a PSII-associated proline-rich protein found in land plants, participates in the maintenance of normal PSII activity under photoinhibitory stress. Here we provide additional evidence for the role of MPH1 in protecting PSII against photooxidative damage. Two Arabidopsis thaliana mutants lacking a functional MPH1 gene suffer from severe photoinhibition relative to the wild-type plants under high irradiance light. The mph1 mutants exhibit significantly decreased PSII quantum yield and electron transport rate after exposure to photoinhibitory light. The mutants also display drastically elevated photodamage to PSII reaction center proteins after high-light treatment. These data add further evidence that MPH1 is involved in PSII photoprotection in Arabidopsis. MPH1 homologs are found across phylogenetically diverse land plants but are not detected in algae or prokaryotes. Taken together, these results suggest that MPH1 protein began to play a role in protecting PSII against excess light following the transition from aquatic to terrestrial conditions.     

The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis

Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling‐lethal. This phenotype was rescued by expressing TerC fused C–terminally to GFP in the terc–1 background, and the resulting terc–1_TerC–GFP line and an artificial miRNA‐based knockdown allele (amiR‐TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR‐TerC plants and terc–1_TerC–GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two‐hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co‐immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR‐TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

Structural changes of the thylakoid membrane network induced by high light stress in plant chloroplasts

Land plants live in a challenging environment dominated by unpredictable changes. A particular problem is fluctuation in sunlight intensity that can cause irreversible damage of components of the photosynthetic apparatus in thylakoid membranes under high light conditions. Although a battery of photoprotective mechanisms minimize damage, photoinhibition of the photosystem II (PSII) complex occurs. Plants have evolved a multi-step PSII repair cycle that allows efficient recovery from photooxidative PSII damage. An important feature of the repair cycle is its subcompartmentalization to stacked grana thylakoids and unstacked thylakoid regions. Thus, understanding the crosstalk between stacked and unstacked thylakoid membranes is essential to understand the PSII repair cycle. This review summarizes recent progress in our understanding of high-light-induced structural changes of the thylakoid membrane system and correlates these changes to the efficiency of the PSII repair cycle. The role of reversible protein phosphorylation for structural alterations is discussed. It turns out that dynamic changes in thylakoid membrane architecture triggered by high light exposure are central for efficient repair of PSII.     

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high‐phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO₂ fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non‐photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton‐motive force across thylakoids. Small‐angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long‐range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild‐type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro‐organization of complexes and induction of photoprotective mechanisms.     

The thylakoid protease Deg1 is involved in photosystem‐II assembly in Arabidopsis thaliana

DegP proteases have been shown to possess both chaperone and protease activities. The proteolytic activities of chloroplast DegP‐like proteases have been well documented. However, whether chloroplast Deg proteases also have chaperone activities has remained unknown. Here we show that chloroplast Deg1 also has chaperone activities, like its Escherichia coli ortholog DegP. Transgenic plants with reduced levels of Deg1 accumulated normal levels of different subunits of the major photosynthetic protein complexes, but their levels of photosystem‐II (PSII) dimers and supercomplexes were reduced. In vivo pulse‐chase protein labeling experiments showed that the assembly of newly synthesized proteins into PSII dimers and supercomplexes was impaired, although the synthesis rate of chloroplast proteins was unaffected in the transgenic lines. Protein overlay assays provided direct evidence that Deg1 interacts with the PSII reaction center protein D2. These results suggest that Deg1 assists the assembly of the PSII complex, probably through interaction with the PSII reaction center D2 protein.     

Light acclimation involves dynamic re‐organization of the pigment–protein megacomplexes in non‐appressed thylakoid domains

Thylakoid energy metabolism is crucial for plant growth, development and acclimation. Non‐appressed thylakoids harbor several high molecular mass pigment–protein megacomplexes that have flexible compositions depending upon the environmental cues. This composition is important for dynamic energy balancing in photosystems (PS) I and II. We analysed the megacomplexes of Arabidopsis wild type (WT) plants and of several thylakoid regulatory mutants. The stn7 mutant, which is defective in phosphorylation of the light‐harvesting complex (LHC) II, possessed a megacomplex composition that was strikingly different from that of the WT. Of the nine megacomplexes in total for the non‐appressed thylakoids, the largest megacomplex in particular was less abundant in the stn7 mutant under standard growth conditions. This megacomplex contains both PSI and PSII and was recently shown to allow energy spillover between PSII and PSI (Nat. Commun., 6, 2015, 6675). The dynamics of the megacomplex composition was addressed by exposing plants to different light conditions prior to thylakoid isolation. The megacomplex pattern in the WT was highly dynamic. Under darkness or far red light it showed low levels of LHCII phosphorylation and resembled the stn7 pattern; under low light, which triggers LHCII phosphorylation, it resembled that of the tap38/pph1 phosphatase mutant. In contrast, solubilization of the entire thylakoid network with dodecyl maltoside, which efficiently solubilizes pigment–protein complexes from all thylakoid compartments, revealed that the pigment–protein composition remained stable despite the changing light conditions or mutations that affected LHCII (de)phosphorylation. We conclude that the composition of pigment–protein megacomplexes specifically in non‐appressed thylakoids undergoes redox‐dependent changes, thus facilitating maintenance of the excitation balance between the two photosystems upon changes in light conditions.     

Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

The plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry‐based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over‐producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.     

The outer membrane Omp85‐like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana

• The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C‐terminal β‐barrel‐shaped membrane domain and one or more N‐terminal polypeptide transport‐associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80.
• We analysed expression of the gene and characterised two T‐DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome.
• We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants.
• We conclude that the function of the plastid‐localised and vein‐specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full‐grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

Arabidopsis OTU1, a linkage‐specific deubiquitinase,is required for endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is part of the ER protein quality‐control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N‐terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N‐terminal region. These observations collectively define AtOTU1 as an OTU domain‐containing deubiquitinase involved in Arabidopsis ERAD.     

Isolation and characterization of a large photosystem I–light‐harvesting complex II supercomplex with an additional Lhca1–a4 dimer in Arabidopsis

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light‐harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1–4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1–a4 dimer bound on the PsaB–PsaI–PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI–LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

Subcellular dynamics of proteins and metabolites under abiotic stress reveal deferred response of the Arabidopsis thaliana hexokinase‐1 mutant gin2‐1 to high light

Stress responses in plants imply spatio‐temporal changes in enzymes and metabolites, including subcellular compartment‐specific re‐allocation processes triggered by sudden changes in environmental parameters. To investigate interactions of primary metabolism with abiotic stress, the gin2‐1 mutant, defective in the sugar sensor hexokinase 1 (HXK1) was compared with its wildtype Landsberg erecta (Ler) based on time resolved, compartment‐specific metabolome and proteome data obtained over a full diurnal cycle. The high light sensitive gin2‐1 mutant was substantially delayed in subcellular re‐distribution of metabolites upon stress, and this correlated with a massive reduction in proteins belonging to the ATP producing electron transport chain under high light, while fewer changes occurred in the cold. In the wildtype, compounds specifically protecting individual compartments could be identified, e.g., maltose and raffinose in plastids, myo‐inositol in mitochondria, but gin2‐1 failed to recruit these substances to the respective compartments, or responded only slowly to high irradiance. No such delay was obtained in the cold. At the whole cell level, concentrations of the amino acids, glycine and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure, and different subcellular allocation of serine may contribute to the slow growth of the gin2‐1 mutant under high irradiance.